CN115054569A - 治疗牙槽骨损伤的dna水凝胶及其制备方法和用途 - Google Patents
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Abstract
本发明提供了一种DNA水凝胶及其制备试剂盒,由序列分别如SEQ IDNO.1~3所示的单链DNA Y1、Y2、Y3自组装而成的Y型DNA单体,与序列分别如SEQ ID NO.4~5所示的单链DNA L1、L2自组装而成的DNA交联剂通过碱基互补配对交联而成。本发明DNA水凝胶可作为IL‑10的有效载体,二者协同增效触发成骨分化、新骨形成,提高骨强度,促进牙槽骨缺损的愈合,在制备治疗牙槽骨损伤的药物中具有很好的应用前景。
Description
技术领域
本发明属于生物医药领域,具体涉及一种治疗牙槽骨损伤的DNA水凝胶及其制备方法和用途。
背景技术
牙槽骨损伤是可能由多种因素导致的牙槽骨完整性遭到破坏的疾病,除了由于不良创伤或外力导致的牙槽骨裂纹、错裂损伤外,牙周炎、根尖病等炎症性疾病引起的骨吸收是导致牙槽骨损伤的重要原因。治疗牙槽骨损伤不但需要促进成骨细胞分化以促进牙槽骨修复,同时还要抑制破骨细胞的活性以减弱骨吸收,因此,存在较大的治疗难度。
特别地,对于糖尿病患者而言,糖尿病牙周炎是糖尿病患者在口腔的主要并发症,也是导致糖尿病患者牙槽骨损伤,最终失牙的重要原因。同时,研究表明高血糖状态下骨缺损愈合时间比健康人更长;一方面,糖尿病高血糖导致巨噬细胞极化异常,会抑制成骨分化,另一方面,大量研究表明,持续高血糖导致骨胶原中糖化终产物增多,以及牙槽骨损伤后炎症反应,会引起伤口周围中性粒细胞和M1型巨噬细胞的大量聚集,释放多种炎症因子,炎症因子表达增多会提高破骨细胞活性,导致骨吸收加剧。因此,糖尿病患者容易发生骨愈合障碍,这也就使得糖尿病患者牙槽骨缺损后更难修复,因此,有效治疗糖尿病患者牙槽骨损伤存在更大的困难。
目前对于糖尿病牙槽骨损伤的治疗例如血糖控制、牙周治疗、抗感染治疗、药物治疗等都未能取得非常好的效果。
细胞因子疗法为糖尿病患者牙槽骨损伤的治疗提供了新思路。细胞因子中IL-10是研究得最好、最广为人知的抗炎细胞因子,IL-10抑制TNF-α、IL-1β和IL-8等细胞因子的表达,并能抑制粘附分子的表达。IL-10能够促使巨噬细胞向M2型转变,M2型巨噬细胞能够促进组织修复和免疫调节,尽管有研究表明IL-10在体外对成骨细胞的促分化和破骨细胞的活性抑制表现出积极的作用,然而,IL-10对于骨愈合障碍的糖尿病病人的牙槽骨损伤治疗效果依然有限。
因此,进一步探索能够有效治疗糖尿病牙槽骨损伤的药物,具有非常重要的意义。
发明内容
本发明的目的在于提供一种治疗牙槽骨损伤的DNA水凝胶及其制备方法和用途。
本发明提供了一种DNA水凝胶制备试剂盒,包括如下单链DNA:
Y1:序列如SEQ ID NO.1所示;
Y2:序列如SEQ ID NO.2所示;
Y3:序列如SEQ ID NO.3所示;
L1:序列如SEQ ID NO.4所示;
L2:序列如SEQ ID NO.5所示。
进一步地,Y1、Y2、Y3、L1和L2的摩尔比为(1~3):(1~3):(1~3):(2~4):(2~4),其中Y1、Y2、Y3等摩尔比,L1、L2等摩尔比;优选地,Y1、Y2、Y3、L1和L2的摩尔比为2:2:2:3:3。
更进一步地,上述Y1、Y2、Y3通过碱基互补配对组装形成Y型DNA单体;L1、L2通过碱基互补配对组装形成DNA交联剂。
进一步地,上述试剂盒还包括细胞因子IL-10,优选地,所述IL-10与Y1的摩尔比为(0.01~0.05):2。
进一步地,它还包括水性溶剂,优选为PBS缓冲液。
本发明还提供了一种DNA水凝胶,它含有Y型DNA单体与DNA交联剂通过碱基互补配对连接形成的交联结构;
所述Y型DNA单体由单链DNA Y1、Y2、Y3通过碱基互补配对组装而成;所述DNA交联剂由单链DNA L1、L2通过碱基互补配对组装而成;
所述单链DNA Y1的序列如SEQ ID NO.1所示,Y2的序列如SEQ ID NO.2所示,Y3的序列如SEQ ID NO.3所示、L1的序列如SEQ ID NO.4所示、L2的序列如SEQ ID NO.5所示;
优选地,所述Y型DNA单体和DNA交联剂的摩尔比为(1~3):(2~4),优选为2:3。
进一步地,它还含有细胞因子IL-10,优选地,所述IL-10与Y型DNA单体的摩尔比为(0.01~0.05):2。
本发明还提供了上述的DNA水凝胶的制备方法,包括如下步骤:
(1)将单链DNA Y1、Y2、Y3溶于水性溶剂,加热到足以使其变性的温度下维持1~3min,再将温度降低到2~8℃孵育,使Y1、Y2、Y3组装形成Y型DNA单体,单体溶液;
将单链DNA L1、L2溶于水性溶剂,加热到足以使其变性的温度下维持1~3min,再将温度降低到2~8℃孵育,使L1、L2组装形成DNA交联剂,得交联剂溶液;
(2)将步骤(1)的单体溶液与交联剂溶液混合,20~30℃孵育制得DNA水凝胶。
进一步地,上述单体溶液和/或交联剂溶液中还溶有细胞因子IL-10。
本发明还提供了上述的DNA水凝胶在制备治疗牙槽骨损伤的药物中的用途;优选地,所述药物是治疗糖尿病患者牙槽骨损伤的药物。
本发明的有益效果:本发明提供了一种由特定序列的DNA单链组装而成的DNA水凝胶,可作为细胞因子药物(例如IL-10)的载体,应用于牙槽骨修复,本发明与细胞因子IL-10复合后的DNA水凝胶,治疗牙槽骨损伤效果优异,显著优于单独使用IL-10、DNA水凝胶的效果,具有协同增效的作用,在制备治疗牙槽骨损伤的药物中具有很好的应用前景。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为本发明DNA水凝胶的合成和及其介导的治疗和修复原理示意图;外源性IL-10刺激M0向M2极化,从而下调促炎因子(TNF-α、MCP-1、IL-6),上调抗炎因子(Arg-1、IL-10),内源性和外源性IL-10均可促进间充质干细胞成骨,促进牙槽骨修复。
图2为本发明DNA水凝胶的合成与表征;其中,A为通过PAGE凝胶电泳表征ssDNA、Y形单体和交联结构单体;B为ILGel溶液态和凝胶态的照片;C为ILGel的流变学分析;D为ILGel的透射电镜图像,白色虚线表示比例尺(比例尺=100纳米);E为ILGel的扫描电镜图像(比例尺=100纳米)。
图3为本发明DNA水凝胶对IL-10的包裹及缓释实验结果;其中,A为DNA水凝胶和ILGel的荧光图像,将用Alexa-488标记的IL-10封装在DNA水凝胶中(比例尺=20μm);B为在指定时间点(0-7天)测定ILGel中IL-10的累积释放量,ILGel在37℃的PBS中持续释放IL-10,用ELISA试剂盒检测释放的IL-10浓度;
图4为本发明DNA水凝胶促进糖尿病牙槽骨损伤修复实验结果;其中,A为PBS、水凝胶、IL-10和ILGel处理后第0天和第21天小鼠下颌骨的代表性micro-ct 3D图像;B为PBS、水凝胶、IL-10、ILGel治疗下颌骨的愈合率;C~F为骨微观结构参数的定量分析,包括BV/TV,Tb.Th,Tb.sp和Tb.N;G为各组小鼠缺损部位的Runx2、ALK-1和MID1免疫组化染色(标尺=50μm);H为免疫组化染色定量检测Runx2、ALK-1和MID1蛋白;
图5为本发明DNA水凝胶在手术后第21天取各组小鼠缺损部位周围组织Runx2(A)、MID1(B)和OPN(C)mRNA水平进行qRT-PCR分析的结果。
具体实施方式
本发明所用原料与设备均为已知产品,通过购买市售产品所得。
实施例1、本发明DNA水凝胶的制备
制备如表1所示的单链DNA。
表1
将Y1、Y2、Y3单链DNA混合在1×PBS(2mM MgCl2,pH=7.2)中,Y1、Y2、Y3的浓度为250mM,将混合溶液从95℃退火到4℃(程序如表2),3条单链DNA自组装形成Y型DNA单体。
将L1、L2单链DNA混合在1×PBS(2mM MgCl2,pH=7.2)中,L1、L2的浓度为250mM,将混合溶液从95℃退火到4℃(程序如表2),2条单链DNA自组装形成DNA交联剂。
表2
通过非变性聚丙烯酰胺凝胶电泳(PAGE)证实了这Y型DNA单体、DNA交联剂的成功合成,如图2A所示。
将得到的Y型DNA单体、DNA交联剂溶液混合(Y形DNA单体与DNA交联剂的摩尔比为2:3,Y形DNA单体的最终浓度为80mM)。混合物在室温下孵育自组装成Hydrogel,混合物的溶液状态立即转化为凝胶状态,制备示意图如图1所示。
实施例2、本发明DNA水凝胶的制备
首先按照实施例1的方法制备得到Y型DNA单体、DNA交联剂的溶液。
然后将细胞因子IL-10(0.5μL的含有0.5μg的IL-10的PBS溶液)加入得到的Y型DNA单体溶液(49.5μL的含有160μM DNA单体的PBS溶液)中混合,将细胞因子IL-10(0.5μL的含有0.5μg的IL-10的PBS溶液)加入得到的DNA交联剂溶液(49.5μL的含有240μM DNA交联剂的PBS溶液)中混合。
将混有IL-10的Y型DNA单体、DNA交联剂的溶液混合(Y形DNA单体与DNA交联剂的摩尔比为2:3,Y形DNA单体的最终浓度为80mM)。混合物在室温下孵育自组装成ILGel,混合物的溶液状态立即转化为凝胶状态,如图2B所示。
以下通过实验例证明本发明的有益效果。
实验例1、本发明DNA水凝胶性能表征
对实施例2制备的ILGel的流变学表征表明ILGel具有典型的水凝胶特性,其中剪切存储模量(G')值高于剪切损失模量(G"),如图2C所示。透射电子显微镜(TEM)图像显示ILGel内部成功形成交联网络,如图2D所示。证明具有交联网络的水凝胶成功合成。进一步使用扫描电子显微镜(SEM)对冻干的ILGel进行表征,观察到ILGel内部有大量多孔的微观结构,如图2E所示,这使得ILGel能够负载或缓释IL-10。
实验例2、本发明DNA水凝胶功能验证
1、为了确认实施例2制备的ILGel中IL-10的成功包被,使用Alexa-488标记IL-10,并进行荧光成像。结果如图3A所示,Alexa-488与DNA水凝胶重叠良好,说明IL-10被DNA水凝胶包裹,进而证明IL-10与DNA水凝胶的成功稳定复合。
2、研究IL-10的释放动力学,将ILGel用PBS浸泡,在指定时间点(0-7天)测定ILGel中IL-10的累积释放量,在指定时间点用ELISA试剂盒检测释放的IL-10浓度。如图3B所示,随着时间的推移,IL-10在PBS中的累积浓度逐渐增加,7d后约有92.31±1.08%的IL-10从ILGel中释放出来,说明ILGel实现了IL-10的长期持续释放。
3、研究ILGel在体内对糖尿病牙槽骨损伤的治疗潜力。用链脲佐菌素(STZ)注射C57BL/6J小鼠诱导1型糖尿病,手术建立上颌牙槽骨缺损模型。分别局部注射PBS、游离IL-10、实施例1的DNA水凝胶(Hydrogel)和实施例2的DNA水凝胶(ILGel)。术后第21天采用micro-ct、免疫组化检测牙槽骨再生情况。
micro-三维重建图像显示,PBS和实施例1的DNA水凝胶治疗组牙槽骨缺陷仍明显,IL-10对缺陷区域的修复作用较显著,实施例2的ILGel修复用极显著。具体的,IL-10给药组愈合率63.30±7.39%,而实施例2的DNA水凝胶ILGel的缺陷愈合率高达93.42±4.6%,显著优于IL-10组(63.30±7.39%),且比游离IL-10和实施例1的DNA水凝胶的效果之和还要更好,说明本发明DNA水凝胶是包裹IL-10用于糖尿病牙槽骨重建的理想生物支架,DNA水凝胶与IL-10复合后,二者协同增效,取得了极显著的治疗牙槽骨损伤的效果。(图4A和B)
4、临床上,小梁结构可作为评估骨强度的独立因素。第21天检测各组小梁结构,包括小梁骨体积(BV/TV)、小梁厚度(Tb.Th)、小梁数量(Tb.N)、小梁分离(Tb.Sp)。
结果如图4C~4F所示,实施例1的DNA水凝胶以及游离IL-10治疗组的BV/TV、Tb和Tb.N相比于PBS组均无明显改善,而实施例2的ILGel治疗组BV/TV、Tb和Tb.N显著提高,且比游离IL-10和实施例1的DNA水凝胶的效果之和还要更好;与PBS组相比,ILGel组牙槽骨缺损部位Tb.Sp显著降低。同时,与PBS或IL-10处理相比,ILGel处理后的牙槽骨缺损中发现Th,说明ILGel处理后的牙槽骨缺损中骨小梁的显微结构再生较好。以上结果进一步说明,DNA水凝胶与IL-10复合后,二者协同增效,提升骨强度。
5、进行免疫组织化学分析和关键成骨的标记分析。结果表明ILGel治疗显著诱导上调Runx2、ALK-1和MID1蛋白,上调缺损区域Runx2、MID1和OPN的mRNA水平(图4G,4H和图5),且上调程度比游离IL-10和实施例1的DNA水凝胶的效果之和还要更高,进一步说明,DNA水凝胶与IL-10复合后,二者协同增效,促进成骨,治疗牙槽骨损伤。本发明DNA水凝胶介导的治疗和修复原理示意图如图1所示。
综上所述,本发明DNA水凝胶可作为IL-10的有效载体,二者协同增效触发成骨分化、新骨形成,提高骨强度,促进牙槽骨缺损的愈合。
SEQUENCE LISTING
<110> 四川大学华西医院
<120> 治疗牙槽骨损伤的DNA水凝胶及其制备方法和用途
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Claims (10)
1.一种DNA水凝胶制备试剂盒,其特征在于,包括如下单链DNA:
Y1:序列如SEQ ID NO.1所示;
Y2:序列如SEQ ID NO.2所示;
Y3:序列如SEQ ID NO.3所示;
L1:序列如SEQ ID NO.4所示;
L2:序列如SEQ ID NO.5所示。
2.如权利要求1所述的试剂盒,其特征在于,Y1、Y2、Y3、L1和L2的摩尔比为(1~3):(1~3):(1~3):(2~4):(2~4),其中Y1、Y2、Y3等摩尔比,L1、L2等摩尔比;优选地,Y1、Y2、Y3、L1和L2的摩尔比为2:2:2:3:3。
3.如权利要求2所述的试剂盒,其特征在于,所述Y1、Y2、Y3通过碱基互补配对组装形成Y型DNA单体;L1、L2通过碱基互补配对组装形成DNA交联剂。
4.如权利要求1所述的试剂盒,其特征在于,它还包括细胞因子IL-10,优选地,所述IL-10与Y1的摩尔比为(0.01~0.05):2。
5.如权利要求1所述的试剂盒,其特征在于,它还包括水性溶剂,优选为PBS缓冲液。
6.一种DNA水凝胶,其特征在于,它含有Y型DNA单体与DNA交联剂通过碱基互补配对连接形成的交联结构;
所述Y型DNA单体由单链DNA Y1、Y2、Y3通过碱基互补配对组装而成;所述DNA交联剂由单链DNA L1、L2通过碱基互补配对组装而成;
所述单链DNA Y1的序列如SEQ ID NO.1所示,Y2的序列如SEQ ID NO.2所示,Y3的序列如SEQ ID NO.3所示、L1的序列如SEQ ID NO.4所示、L2的序列如SEQ ID NO.5所示;
优选地,所述Y型DNA单体和DNA交联剂的摩尔比为(1~3):(2~4),更优选为2:3。
7.如权利要求6所述的水凝胶,其特征在于,它还含有细胞因子IL-10,优选地,所述IL-10与Y型DNA单体的摩尔比为(0.01~0.05):2。
8.如权利要求6或7所述的DNA水凝胶的制备方法,其特征在于,包括如下步骤:
(1)将单链DNA Y1、Y2、Y3溶于水性溶剂,加热到足以使其变性的温度下维持1~3min,再将温度降低到2~8℃孵育,使Y1、Y2、Y3组装形成Y型DNA单体,单体溶液;
将单链DNA L1、L2溶于水性溶剂,加热到足以使其变性的温度下维持1~3min,再将温度降低到2~8℃孵育,使L1、L2组装形成DNA交联剂,得交联剂溶液;
(2)将步骤(1)的单体溶液与交联剂溶液混合,20~30℃孵育制得DNA水凝胶。
9.如权利要求8所述的制备方法,其特征在于,所述单体溶液和/或交联剂溶液中还溶有细胞因子IL-10。
10.权利要求6或7所述的DNA水凝胶在制备治疗牙槽骨损伤的药物中的用途;优选地,所述药物是治疗糖尿病患者牙槽骨损伤的药物。
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